Marzena Warchoł

Application of chosen factors in the wide crossingmethod for the production of oat doubled haploids

Abstract Oat (Avena sativa L.) has recently gained importance due to the discovery of a variety of health benefits and new opportunities of use. There is no efficient protocol for the production of oat doubled haploid (DH) lines. The aim of this study was to increase the efficiency of obtaining DHs of oat by the wide crossing method. The study was performed on five oat genotypes. We have compared the induction of embryos after pollination with maize, sorghum and millet pollen as well as the development of haploid embryos isolated 2, 3 and 4 weeks after pollination and cultivated on media with different sugar content. Haploid plants were treated with colchicine after or before acclimation to natural conditions. Of the three types of pollen used, the largest number of haploid embryos was obtained using maize pollen. Three weeks after pollination was the most suitable time for the isolation and cultivation of the embryos. The most efficient medium enabling the development of embryos and conversion to plants was 190-2 containing 9% of maltose. Colchicine treatment of acclimated plants provided high survival rate.

The effect of light intensity on the production of oat (Avena sativa L.) doubled haploids through oat × maize crosses

Oat haploid embryos were obtained by wide crossing with maize. The effect of light intensity during the growing period of donor plants (450 and 800 μmol m−2 s−1) and in vitro cultures (20, 40, 70 and 110 μmol m−2 s−1) was examined for the induction and development of oat DH lines. Oat florets (26008) from 32 genotypes were pollinated with maize and treated with 2,4-dichlorophenoxyacetic acid. All the tested genotypes formed more haploid embryos when donor plants were grown in a greenhouse (9.4%) compared to a growth chamber (6.1%). The light intensity of 110 μmol m−2 s−1 during in vitro culture resulted in the highest percentage of embryo germination (38.9%), conversion into plants (36.4%) and DH line production (9.2%) when compared with lower light intensities (20, 40 and 70 μmol m−2 s−1). The results show that the growth conditions of the donor plant and light intensity during in vitro culture can affect the development of haploid embryos. This fact may have an impact on oat breeding programs using oat × maize crosses.

Biochemical and morphometric analysis of Rosa tomentosa and Rosa rubiginosa during application of liquid culture systems for in vitro shoot production

ABSTRACT Two liquid culture systems, rotary shaker (RS) and temporary immersion system (TIS) for the propagation of Rosa rubiginosa and Rosa tomentosa were investigated and compared with solid medium culture. Shoot tips were grown on basic Murashige and Skoog medium with 20 mg dm−3 Fe EDDHA, 1 µM BA, 1.5 µM GA3 and 3% sucrose for six weeks. Of the different culture systems, liquid cultures resulted in better growth and development of roses. Both RS and TIS cultures improved biomass growth, multiplication rate, leaf blade area and plant height; however, RS reduced the shoot dry mass content. There were no changes in the content of chlorophyll a, b, a + b in plants developed in the tested systems. Use of a solid medium favoured the accumulation of phenolic compounds, soluble sugars, and carotenoids, but it was accompanied by browning and necrosis of shoots. Plants propagated in TIS were characterised by the high content of phenolic compounds and soluble sugars, but they had the highest multiplication rate. Use of TIS resulted in eight times the number of shoots for R. tomentosa and twice as many for R. rubiginosa compared to solid medium.

Complex characterization of oat (Avena sativa L.) lines obtained by wide crossing with maize (Zea mays L.)

Background The oat × maize addition (OMA) lines are used for mapping of the maize genome, the studies of centromere-specific histone (CENH3), gene expression, meiotic chromosome behavior and also for introducing maize C4 photosynthetic system to oat. The aim of our study was the identification and molecular-cytogenetic characterization of oat × maize hybrids. Methods Oat DH lines and oat × maize hybrids were obtained using the wide crossing of Avena sativa L. with Zea mays L. The plants identified as having a Grande-1 retrotransposon fragment, which produced seeds, were used for genomic in situ hybridization (GISH). Results A total of 138 oat lines obtained by crossing of 2,314 oat plants from 80 genotypes with maize cv. Waza were tested for the presence of maize chromosomes. The presence of maize chromatin was indicated in 66 lines by amplification of the PCR product (500 bp) generated using primers specific for the maize retrotransposon Grande-1. Genomic in situ hybridization (GISH) detected whole maize chromosomes in eight lines (40%). All of the analyzed plants possessed full complement of oat chromosomes. The number of maize chromosomes differed between the OMA lines. Four OMA lines possessed two maize chromosomes similar in size, three OMA—one maize chromosome, and one OMA—four maize chromosomes. In most of the lines, the detected chromosomes were labeled uniformly. The presence of six 45S rDNA loci was detected in oat chromosomes, but none of the added maize chromosomes in any of the lines carried 45S rDNA locus. Twenty of the analyzed lines did not possess whole maize chromosomes, but the introgression of maize chromatin in the oat chromosomes. Five of 66 hybrids were shorter in height, grassy type without panicles. Twenty-seven OMA lines were fertile and produced seeds ranging in number from 1–102 (in total 613). Sixty-three fertile DH lines, out of 72 which did not have an addition of maize chromosomes or chromatin, produced seeds in the range of 1–343 (in total 3,758). Obtained DH and OMA lines were fertile and produced seeds. Discussion In wide hybridization of oat with maize, the complete or incomplete chromosomes elimination of maize occur. Hybrids of oat and maize had a complete set of oat chromosomes without maize chromosomes, and a complete set of oat chromosomes with one to four retained maize chromosomes.

Melatonin significantly influences seed germination and seedling growth of Stevia rebaudiana Bertoni

Background Melatonin (MEL) is a signaling molecule in plants that affects developmental processes during vegetative and reproductive growth. Investigations have proved that exogenously applied MEL also has the potential to improve seed germination and plant development. Methods In the present study, seeds of stevia, a species with a very low germination rate, were germinated on an agar gel (AG) containing MEL at various concentrations (5, 20, 100, and 500 µM) in light. Seeds germinated on AG without MEL were used as controls. For the first 24 or 48 h of germination, the seeds were maintained in darkness as a pre-incubation step. Some seeds were not exposed to this pre-incubation step. Results At concentrations of 20 and 5 µM, MEL significantly improved germination, but only in seeds pre-incubated in darkness for 24 h (p < 0.001). At concentrations of 100 and 500 µM, MEL had an inhibitory effect on germination, regardless of the pre-incubation time. Melatonin also affected plantlet properties. At a concentration of 20 µM, MEL increased plantlet fresh weight and leaf numbers. At a concentration of 5 µM, it promoted plantlet height. Regarding root development, the most favorable MEL concentration was 500 µM. Biochemical analysis revealed that MEL promoted higher pigment concentrations but hampered superoxide dismutase activity. On the other hand, the concentrations of sugars and phenolics, as well as the activities of catalase and peroxidase, increased at a MEL concentration of 500 µM. Discussion The results suggest that MEL can improve germination of positively photoblastic stevia seeds and that it can play a role in plantlet development. However, the effects observed in the present study depended on the quantity of MEL that was applied.

Obtaining of winter rye (Secale cereale L. ssp. cereale) haploid embryos through hybridization with maize (Zea Mays L.)

The aim of this study was to determine the effect of selected factors on rye (Secale cereale L.) haploid embryo production by the wide crossing method. The study was performed on fifteen winter rye genotypes. This is the first time for rye when besides the genotype, on the enlargement of ovaries and haploid embryo production, such factors as: type of auxin analogues 2,4-dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram), and the time between florets emasculation and pollination were investigated. All factors had a significant impact on rye ovary enlargement, however the haploid embryo formation depended only on rye genotype, not on kind of auxin and days between emasculation to pollination. In total, twenty one haploid embryos were formed by six genotypes of fifteen tested. On average, 13.86% (after 2,4-D treatment) to 20.05% (after dicamba treatment) enlarged ovaries per emasculated florets were obtained. Most of the ovaries enlarged when florets were pollinated 4 and 6 days after emasculation. Most of the haploid embryos formed when florets were pollinated 6 days after emasculation. The obtained haploid embryos did not germinate.

The effect of genotype, media composition, pH and sugar concentrations on oat (Avena sativa L.) doubled haploid production through oat × maize crosses

Doubled haploid (DH) technology in oat has not reached the same stage as in other cereals leading to its application in plant breeding. The objective of this investigation was to increase the effectiveness of Avena sativa L. haploid embryo germination obtained by the distant crosses with maize. Developed embryos (obtained from 22 genotypes) were transferred on five germination media: MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) with 3% sucrose, pH 5.8 (control medium), and 190-2 supplemented with 6 and 9% maltose. The pH of 190-2 was adjusted to 5.5 and 6.0. Of all tested genotypes, 591 haploid embryos were obtained, almost half of them (279) germinated. The rate of haploid embryo germination induced on 190-2 was 6.92%, while in MS it was 3.25%. The sugar and its concentration significantly affected the germination of haploid embryos. The highest percentage of haploid embryo germination (9.11%) and DH lines production (1.64%) was achieved on 190-2 with 9% maltose and pH 6.0. All DH lines are incorporated to breeding programs for the development of new cultivars.

Chlorophyll a fluorescence parameters in the evaluation of oat DH lines yield components

Chlorophyll a fluorescence can provide insight into the ability of plants to tolerate environmental conditions that can damage photosynthetic apparatus and decrease yield. The aim of the study was to determine the relationship between chlorophyll a fluorescence parameters and yield components of oat DH lines. All DH lines significantly differed in chlorophyll a fluorescence parameters and yield components. The overall performance index of PSII photochemistry (PI), showed the highest variation between DH lines, whereas the lowest had the ratio of variable to maximum fluorescence (Fv/Fm). The highest differences were observed in the number of grains per plant (21.3 to 600). Thousand-grain weight varied from 17.82 g to 41.01 g and the biomass from 8.01 g to 29.31 g. The highest negative correlations were found between Fv/Fm, Area (pool size of electron acceptors from PSII), PI and grain number per plant and biomass. Positive correlations were observed between light energy absorption (ABS/CS), grain number per plant and biomass, as well as the amount of excitation energy trapped in PSII reaction centers (TRo/CS) and biomass. Principal component analysis of chlorophyll a fluorescence parameters, together with yield components, discriminated two oat DH lines groups according to their photosynthetic efficiency and yield.